Regulation of gene expression by noncoding RNAs (ncRNAs) is an emerging paradigm in biology. ncRNAs regulate a variety of processes, including transcription, translation, RNA modification, mRNA stability, RNA splicing, chromatin structure, and protein stability and activity, in a wide variety of organisms from bacteriophages to humans. Eukaryotic and prokaryotic genomes contain hundreds of uncharacterized ncRNAs, and these genes have been increasingly identified as the loci of mutations that cause developmental defects and several human diseases. One of the most interesting of these ncRNAs is tmRNA, a molecule with properties of both a tRNA and an mRNA, which intervenes in selected translation reactions, tmRNA recognizes selected translation complexes and enters the ribosome to mediate the addition of a peptide tag to the nascent polypeptide, targeting the protein for rapid degradation and releasing the ribosome and mRNA. This unique activity functions both in a translational quality control mechanism and in regulation of gene expression. tmRNA is conserved throughout the bacterial kingdom, and is required for processes such as pathogenesis, symbiosis, stress tolerance, and bacterial development. The long-term objective of this proposal is to understand the mechanism of action and physiological role of tmRNA in bacteria. This proposal focuses on the role of tmRNA in the development and cell cycle regulation of Caulobacter crescentus. C. crescentus is the organism of choice for these studies because the extensive knowledge of the molecular events that control its cell cycle and developmental program provide a unique opportunity to determine the influence of tmRNA activity on cellular differentiation and DNA replication. tmRNA is required for proper timing of DNA replication and the coincident differentiation from swarmer cell to stalked cell in C. crescentus. The primary goals of this proposal are: to identify substrates for tmRNA in C. crescentus that are important for control of DNA replication, to determine how tmRNA activity on these substrates affects DNA replication, and to understand how the activity of tmRNA is controlled by the genetic regulatory network of the cell. [unreadable] [unreadable]